This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptides of the present invention are human chemotactic cytokine I polypeptides. The invention also relates to inhibiting the action of such polypeptides.
The cytokine family of proteins exhibits a wide variety of functions. A hallmark feature is their ability to elicit chemotactic migration of distinct cell types, including polymorphonuclear cells and macrophages. Many cytokines have proinflammatory activity and are involved in multiple steps during inflammatory reactions. In addition to their involvement in inflammation, cytokines have been shown to exhibit other activities. For example, interleukin-8 (IL-8) promotes proliferation of keratinocytes.
In light of the diverse biological activities, it is not surprising that cytokines have been implicated in a number of physiological and disease conditions, including lymphocyte trafficking, wound healing, hematopoietic regulation and immunological disorders such as allergy, asthma and arthritis.
The S100 family of calcium binding proteins has chemotactic activity for polymorphonuclear cells, mononuclear cells and neutrophils and are calcium binding proteins. The S100 protein has been recently identified in cells of myeloid origin and consists of macrophage inhibitory factor-related protein (MRP-8), MRP-14, chemotactic protein 10 (CP-10) and calgranulin C.
MRP-8 and MRP-14 belong to the S100 family of proteins, which includes calbindin, and calcyclin (Kligman, D. and Hilt, D. C., Trends Biochem. Sci., 13:437-443 (1998)). This group of protein ranges in molecular size from 10 to 14 kd and are also expressed in a cell lineage-specific manner. Alignment of individual sequences shows that there is overall conservation of structure within the family, a notable feature being the two calcium binding sites, which are the “EF hand” type. Sequences at both the NH2- and COOH-terminal ends of MRP-8 and MRP-14 are relatively hydrophobic. An attractive hypothesis is that these regions of the molecule are buried until calcium binding brings about the conformational changes that cause their exposure, making them potentially available for interactions with other effector molecules. Because of the extended sequence of its COOH-terminal “tail” MRP-14 is the largest member of the S100 family. (Hessian, P., et al., J. Leuk. Bio., 53:197-204 (1993).
Each gene in the S100 family is composed of three exons with one intron interrupting the protein-coding sequence between the two EF hands. The MRP-8 and MRP-14 genes are both localized to chromosome 1Q12-Q21 with an undefined distance between them (Dorin J. R., et al., Nature, 326:614-617 (1987) and Lagasse, E. and Clerc, R. G., Mol. Cell. Biol., 8:2402, 2410 (1988)). Two other S100 family members 1882 (CAPL) and calcyclin/2A9 (CACY) also map to chromosome 1Q12-Q21 (Dorian, J. R., et al., Genomics, 8:420-426 (1990). It is probably that co-segregation of these five genes on chromosome 1 may represent an S100 family locus. However, this does not apply to all S100 homologs. MRP-8 and MRP-14 are restricted to cells of the monocytes/macrophage lineage, neutrophils, and under certain circumstance keratinocytes, suggesting that its expression is tightly regulated during differentiation (Hogg, N., et al., Eur. J. Immunol., 19:1053-1061 (1989)). Thus, monocytes and neutrophils in the circulation express MRP-8 and MRP-14, in contrast to other related cells such as lymphocytes, platelets, basophils and eocynophils which do not (Id.).
Resident tissue macrophages do not express MRP-8 and MRP-14, implying that differentiation of monocytes to macrophages is normally associated with loss of this molecule (Id.). Furthermore, immunohistochemical data show that at inflammatory sites MRP-8 and MRP-14 positive cells are associated with vessels but that the majority of monocytes already within the tissues at these sites have lost MRP-8 and MRP-14 expression (Id.). In keeping with these observations, tissue culture-matured monocytes down regulate this molecule (Zwadlo, G., et al., Clin. Exp. Immunol., 72:510-515 (1988)).
At sites of chronic inflammation in patients with diseases such as rheumatoid arthritis, sarcoidosis, tuberculosis or onchocerciasis macrophages express both MRP-8 and MRP-14 (Palmer, D. G., et al., Clin. Immunol. Immunopathol., 45:17-28 (1987)). In contrast, macrophages in acutely inflamed tissues may express only MRP-14 (Delabie, J., et al., Clin. Exp. Immunol., 81:123-126 (1990)). The expression of MRP-8 and MRP-14 by macrophages could be flecked exposure to tissue stimuli that either maintain expression or induce re-expression of the molecule (Palmer, D. G., et al., Clin. Immunol. Immunopathol., 45:17-28 (1987)).
In common with other members of the S100 family, MRP-8 and MRP-14 are found predominately in a cytosolic location in both monocytes and neutrophils (Dale, I., et al., Eur. J. Biochem., 134:1-6 (1983)). It is also possible that MRP-8 and MRP-14 can be expressed on the cell surface, although the majority of antibodies specific for these proteins do not react with circulating monocytes or neutrophils. There is also evidence that MRP-8 and MRP-14 exist extracellularly, however, neither protein has the signal peptide sequence for membrane translocation. Thus, MRP-8 and MRP-14 fall into the category of proteins, including interleukin-1 and basic fibroblast growth factor, that clearly have extracellular functions but about which little is known of their cellular release. Finally, MRP-8 and MRP-14 are found in the serum of patients with cystic fibrosis and other chronic inflammatory states such as rheumatoid arthritis and sarcoidosis (Bullock, S., et al., Clin. Genet., 21:336-341 (1982)).
CP-10 is one of the most potent chemotactic proteins of the S100 family. An extracellular function of the murine CP-10 includes a potent chemotactic agent involved in phagocyte recruitment during inflammatory reactions (Lackman, M., et al., J. Biol. Chem., 267:7499 (1992)). CP-10 has an apparent molecular weight of 10.3 kd and a complete sequence of 88 amino acids.
S100 proteins are characterized by two calcium binding regions, which are strongly conserved and are separated by an 8 to 12 amino acid hinge region (Kligman, D., Trends Biochem. Sci., 13:437 (1988)). Although the hinge region length is conserved, the amino acid sequences are widely divergent. This divergence led to the hypothesis that the hinge region may concur functional specificity by interaction with the actor proteins (Id.).